Peroxisomes are ubiquitous eukaryotic organelles involved in a variety of important metabolic pathways. In humans, peroxisomes are essential for the metabolism of certain lipids, and defects in their biogenesis are responsible for a family of lethal genetic disorders termed peroxisome biogenesis disorders (PBDs). The primary long-term goal of this program is to understand, at the molecular level, the mechanisms controlling peroxisomal biogenesis. A combined molecular genetic and biochemical attack has been initiated using the yeast Pichia pastoris as the model system. This yeast is unique in that peroxisomes are massively and specifically induced in size and number in response to either of two carbon sources, methanol or oleic acid. Primarily through the isolation ofpex mutants in P. pastoris and other yeasts, 25 different PEX genes have been identified and their products, (peroxins or PEX proteins) have been described. It is likely that many other genes are involved in peroxisome biogenesis but cannot be identified by the genetic approach. We propose to take advantage of the recent availability of the P. pastoris genomic sequence and test the feasibility of identifying new PEX genes via a gene profiling approach. In this application, we propose to construct microarrays containing a selected subset of P. pastoris genes and utilize the arrays to examine changes in transcription that occur in response to methanol and oleic acid. In particular, we will focus on the development of physiological (chemostat culturing) and genetic (certain mutant backgrounds) methods to further enhance the methanol- and oleate-induced transcriptional responses in P. pastoris. Genes that are strongly induced in response to either or both of these carbon sources will be identified as PEXgene candidates. P. pastoris strains deleted for each strain will be constructed and examined biochemically and morphologically for peroxisome function to identify new PEX genes and to define their role in peroxisome biogenesis.